[Show abstract][Hide abstract] ABSTRACT: The relative localization patterns of class B penicillin-binding proteins Pbp2x and Pbp2b were used as positional indicators of septal and peripheral (side-wall-like) peptidoglycan (PG) synthesis, respectively, in the midcell regions of Streptococcus pneumoniae cells at different stages of division. We confirm that Pbp2x and Pbp2b are essential in the strain D39 genetic background, which differs from that of laboratory strains. We show that Pbp2b, like Pbp2x and class A Pbp1a, follows a different localization pattern than FtsZ and remains at division septa after FtsZ reappears at the equators of daughter cells. Pulse-experiments with fluorescent D-amino acids (FDAAs) were performed in wild-type cells and in cells in which Pbp2x activity was preferentially inhibited by methicillin or Pbp2x amount was depleted. These experiments show that Pbp2x activity separates from that of other PBPs to the centers of constricting septa in mid-to-late divisional cells resolved by high-resolution 3D-SIM microscopy. Dual-protein and protein-fluorescent vancomycin 2D and 3D-SIM immunofluorescence microscopy (IFM) of cells at different division stages corroborate that Pbp2x separates to the centers of septa surrounded by an adjacent constricting ring containing Pbp2b, Pbp1a, and regulators, StkP and MreC. The separate localization of Pbp2x suggests distinctive roles in completing septal PG synthesis and remodeling.
[Show abstract][Hide abstract] ABSTRACT: The interphase microtubule arrays in flowering plant cells assemble at the cell cortex into patterns that affect cellular morphogenesis. A decade of live cell imaging studies has provided significant information about the in vivo properties of the microtubule polymers. Efforts to extrapolate individual properties to larger roles in organizing or patterning the microtubule array have produced models focused on self-organization and local levels of biological control. Recent studies looking at cortical microtubule arrays as they transition from an existing pattern to a new pattern have re-emerged as a testbed for examining these models and the molecular hypotheses underpinning them. The evidence suggests that microtubule patterning is locally controlled on the scale of a cell face, using or circumventing self-organizating properties as necessary.
Current opinion in plant biology 12/2013; 16(6):693-7. · 10.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proper spindle assembly and chromosome segregation rely on precise microtubule dynamics, which are governed in part by the kinesin-13 MCAK. MCAK microtubule depolymerization activity is inhibited by Aurora B-dependent phosphorylation, but the mechanism of this inhibition is not understood.
Here, we develop the first Förster resonance energy transfer (FRET)-based biosensor for MCAK and show that MCAK in solution exists in a closed conformation mediated by an interaction between the C-terminal domain (CT) and the neck. Using fluorescence lifetime imaging (FLIM) we show that MCAK bound to microtubule ends is closed relative to MCAK associated with the microtubule lattice. Aurora B phosphorylation at S196 in the neck opens MCAK conformation and diminishes the interaction between the CT and the neck. Using FLIM and TIRF imaging, we find that changes in MCAK conformation are associated with a decrease in MCAK affinity for the microtubule.
Unlike motile kinesins, which are open when doing work, the high-affinity binding state for microtubule-depolymerizing kinesins is in a closed conformation. Phosphorylation switches MCAK conformation, which inhibits its ability to interact with microtubules and reduces its microtubule depolymerization activity. This work shows that the conformational model proposed for regulating kinesin activity is not universal and that microtubule-depolymerizing kinesins utilize a distinct conformational mode to regulate affinity for the microtubule, thus controlling their catalytic efficiency. Furthermore, our work provides a mechanism by which the robust microtubule depolymerization activity of kinesin-13s can be rapidly modulated to control cellular microtubule dynamics.
Current biology: CB 11/2013; · 10.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacterial cell shapes are manifestations of programs carried out by multi-protein machines that synthesize and remodel the resilient peptidoglycan mesh and other polymers surrounding cells. GpsB protein is conserved in low-GC Gram-positive bacteria and is not essential in rod-shaped Bacillus subtilis, where it plays a role in shuttling penicillin binding proteins (PBPs) between septal and side-wall sites of peptidoglycan (PG) synthesis. In contrast, we report here that GpsB is essential in ellipsoid-shaped, ovococcal Streptococcus pneumoniae (pneumococcus), and depletion of GpsB leads to formation of elongated, enlarged cells containing unsegregated nucleoids and multiple, unconstricted rings of new peptidoglycan (PG), and eventual lysis. These phenotypes are similar to those caused by selective inhibition of Pbp2x by methicillin that prevents septal PG synthesis. Dual-protein 2D and 3D-SIM (structured illumination) immunofluorescence microscopy (IFM) showed that GpsB and FtsZ have overlapping, but not identical, patterns of localization during cell division and that multiple, unconstricted rings of division proteins FtsZ, Pbp2x, Pbp1a, and MreC are in elongated cells depleted of GpsB. These patterns suggest that GpsB, like Pbp2x, mediates septal ring closure. This first dual-protein 3D-SIM IFM analysis also revealed separate positioning of Pbp2x and Pbp1a in constricting septa, consistent with two separable PG synthesis machines.
[Show abstract][Hide abstract] ABSTRACT: The advent of fluorescent proteins and access to modern imaging technologies have dramatically accelerated the pace of discovery in plant cell biology. Remarkable new insights into such diverse areas as plant pathogenesis, cytoskeletal dynamics, sugar transport, cell wall synthesis, secretory control, and hormone signaling have come from careful examination of living cells using advanced optical probes. New technologies, both commercially available and on the horizon, promise a continued march toward more quantitative methods for imaging and for extending the optical exploration of biological structure and activity to molecular scales. In this review, we lay out fundamental issues in imaging plant specimens and look ahead to several technological innovations in molecular tools, instrumentation, imaging methods, and specimen handling that show promise for shaping the coming era of plant cell biology. Expected final online publication date for the Annual Review of Plant Biology Volume 64 is April 29, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Annual Review of Plant Biology 03/2013; · 18.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The acentriolar cortical microtubule arrays in dark-grown hypocotyl cells organize into a transverse coaligned pattern that is critical for axial plant growth. In light-grown Arabidopsis thaliana seedlings, the cortical array on the outer (periclinal) cell face creates a variety of array patterns with a significant bias (>3:1) for microtubules polymerizing edge-ward and into the side (anticlinal) faces of the cell. To study the mechanisms required for creating the transverse coalignment, we developed a dual-hormone protocol that synchronously induces ∼80% of the light-grown hypocotyl cells to form transverse arrays over a 2-h period. Repatterning occurred in two phases, beginning with an initial 30 to 40% decrease in polymerizing plus ends prior to visible changes in the array pattern. Transverse organization initiated at the cell's midzone by 45 min after induction and progressed bidirectionally toward the apical and basal ends of the cell. Reorganization corrected the edge-ward bias in polymerization and proceeded without transiting through an obligate intermediate pattern. Quantitative comparisons of uninduced and induced microtubule arrays showed a limited deconstruction of the initial periclinal array followed by a progressive array reorganization to transverse coordinated between the anticlinal and periclinal cell faces.
[Show abstract][Hide abstract] ABSTRACT: Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function, and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labeling flagellar basal bodies, hooks, and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by the protein of unknown function SwrA. Basal bodies are assembled rapidly (< 5 minutes) but the assembly of flagella capable of supporting motility (> 40 minutes) is rate limited by filament polymerization. We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid-like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologs to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella in individual cells than the motile behavior of populations.
[Show abstract][Hide abstract] ABSTRACT: We investigated the role of the Arabidopsis microtubule associated proteins 65-1 and 65-2 (MAP65-1 and MAP65-2) in the control of axial root growth. Transgenic plants expressing fluorescent fusion proteins from native promoters indicated exactly overlapping accumulation of MAP65-1 and MAP65-2 in the root tip and elongation zone. Nearly identical protein accumulation patterns were observed when MAP65-1 and MAP65-2 were expressed behind a constitutive CaMV 35S promoter, suggesting a level of post-transcriptional control that restricts these proteins to rapidly growing portions of the root. Co-expression of MAP65-1 and MAP65-2 fusion proteins showed precise co-localization to interphase and cytokinetic microtubule arrays. In interphase root tip cells, the fluorescent protein fusions labeled microtubules that were organized into a variety of different array patterns. In the rapidly growing cells of the root elongation zone, we found MAP65-1 and MAP65-2 co-localized exclusively to the lateral faces of cells that were axially extending. Genetic analysis showed that MAP65-1 and MAP65-2 are coordinately required for proper root elongation. Double map65-1-1 map65-2-2 mutant roots from dark-grown plants contained 50% fewer cells per file than wild-type roots, but we found no evidence that cytokinesis was disrupted. We additionally discovered that cell length was significantly shorter in the mature regions of the root beyond the zone where MAP65-1 and MAP65-2 accumulated. Our data indicate that MAP65-1 and MAP65-2 play a critical role in root growth by promoting cell proliferation and axial extension.
The Plant Journal 03/2012; 71(3):454-63. · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microtubule polymers typically function through their collective organization into a patterned array. The formation of the pattern, whether it is a relatively simple astral array or a highly complex mitotic spindle, relies on controlled microtubule nucleation and the basal dynamics parameters governing polymer growth and shortening. We have investigated the interaction between the microtubule nucleation and dynamics parameters, using macroscopic Monte Carlo simulations, to determine how these parameters contribute to the underlying microtubule array morphology (i.e. polymer density and length distribution). In addition to the well-characterized steady state achieved between free tubulin subunits and microtubule polymer, we propose that microtubule nucleation and extinction constitute a second, interdependent steady state process. Our simulation studies show that the magnitude of both nucleation and extinction additively impacts the final steady state free subunit concentration. We systematically varied individual microtubule dynamics parameters to survey the effects on array morphology and find specific sensitivity to perturbations of catastrophe frequency. Altering the cellular context for the microtubule array, we find that nucleation template number plays a defining role in shaping the microtubule length distribution and polymer density.
Computational biology and chemistry 10/2011; 35(5):269-81. · 1.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mitotic spindle is a macromolecular structure utilized to properly align and segregate sister chromatids to two daughter cells. During mitosis, the spindle maintains a constant length, even though the spindle microtubules (MTs) are constantly undergoing polymerization and depolymerization . Members of the kinesin-8 family are important for the regulation of spindle length and for chromosome positioning [2-9]. Kinesin-8 proteins are length-specific, plus-end-directed motors that are proposed to be either MT depolymerases [3, 4, 8, 10, 11] or MT capping proteins . How Kif18A uses its destabilization activity to control spindle morphology is not known. We found that Kif18A controls spindle length independently of its role in chromosome positioning. The ability of Kif18A to control spindle length is mediated by an ATP-independent MT binding site at the C-terminal end of the Kif18A tail that has a strong affinity for MTs in vitro and in cells. We used computational modeling to ask how modulating the motility or binding properties of Kif18A would affect its activity. Our modeling predicts that both fast motility and a low off rate from the MT end are important for Kif18A function. In addition, our studies provide new insight into how depolymerizing and capping enzymes can lead to MT destabilization.
Current biology: CB 08/2011; 21(17):1500-6. · 10.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis thaliana MAP65-1 and MAP65-2 genes are members of the larger eukaryotic MAP65/ASE1/PRC gene family of microtubule-associated proteins. We created fluorescent protein fusions driven by native promoters that colocalized MAP65-1 and MAP65-2 to a subset of interphase microtubule bundles in all epidermal hypocotyl cells. MAP65-1 and MAP65-2 labeling was highly dynamic within microtubule bundles, showing episodes of linear extension and retraction coincident with microtubule growth and shortening. Dynamic colocalization of MAP65-1/2 with polymerizing microtubules provides in vivo evidence that plant cortical microtubules bundle through a microtubule-microtubule templating mechanism. Analysis of etiolated hypocotyl length in map65-1 and map65-2 mutants revealed a critical role for MAP65-2 in modulating axial cell growth. Double map65-1 map65-2 mutants showed significant growth retardation with no obvious cell swelling, twisting, or morphological defects. Surprisingly, interphase microtubules formed coaligned arrays transverse to the plant growth axis in dark-grown and GA(4)-treated light-grown map65-1 map65-2 mutant plants. We conclude that MAP65-1 and MAP65-2 play a critical role in the microtubule-dependent mechanism for specifying axial cell growth in the expanding hypocotyl, independent of any mechanical role in microtubule array organization.
The Plant Cell 05/2011; 23(5):1889-903. · 9.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We generalize the Dogterom-Leibler model for microtubule dynamics (Dogterom and Leibler in Phys Rev Lett 70(9):1347-1350, 1993) to the case where the rates of elongation as well as the lifetimes of the elongating shortening phases are a function of GTP-tubulin concentration. We analyze also the effect of nucleation rate in the form of a damping term which leads to new steady-states. For this model, we study existence and stability of steady states satisfying the boundary conditions at x=0. Our stability analysis introduces numerical and analytical Evans function computations as a new mathematical tool in the study of microtubule dynamics.
Journal of Mathematical Biology 11/2010; 63(3):459-92. · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We tested the general hypothesis that bundling stabilizes the dynamic properties of the constituent microtubules (MTs) in vivo. We quantified the assembly dynamics of bundled and unbundled MTs in the interphase cortical array of Arabidopsis hypocotyl cells using high dynamic range spinning disk confocal microscopy. We find no evidence that bundled MTs are stabilized against depolymerization through changes to their dynamic properties. Our observations of MT plus and minus ends indicate that both bundled and unbundled polymers undergo persistent treadmilling in this system. We conclude that the temporal persistence of MT subassemblies in the Arabidopsis cortical array is largely dependent upon recruitment or nucleation of new treadmilling MTs and not on polymer stabilization. Monte Carlo simulations suggest that small differences discovered in the dynamic properties between bundled and unbundled polymers would produce relatively small macroscopic effects on the larger MT array.
[Show abstract][Hide abstract] ABSTRACT: Microtubules assemble into arrays of bundled filaments that are critical for multiple steps in cell division, including anaphase and cytokinesis. Recent structural and functional studies, including two papers in this issue of Cell (Bieling et al., 2010; Subramanian et al., 2010), demonstrate how the MAP65 protein PRC1 crosslinks microtubules and cooperates with kinesin motors to control the dynamics and size of bundled regions.
[Show abstract][Hide abstract] ABSTRACT: Template matching is a common approach for identifying fluorescent objects within a biological image. But how to decide a threshold value for the purpose of justifying the goodness of matching score is a rather difficult task. In this paper, we propose a framework that dynamically chooses appropriate threshold values for correct object identification at a non-arbitrary statistical power based on the local measure of signal and noise. We validate the feasibility of our proposed framework by presenting simulation experiments conducted with both synthetic and live-cell data sets. The experimental results suggest that our auto-thresholding algorithm and local signal to noise ratio estimation can provide solid means for effective spot identity in place of an ad hoc threshold fitting value or minimization method.
20th International Conference on Pattern Recognition, ICPR 2010, Istanbul, Turkey, 23-26 August 2010; 01/2010
[Show abstract][Hide abstract] ABSTRACT: Microtubules (MTs) are highly dynamic polymers that serve as tracks for vesicular movement during interphase and as structural components of the mitotic spindle, which is used to segregate the genetic material. MT dynamics are highly regulated wherein MTs turnover differentially between interphase and mitosis. Within the mitotic spindle, there are distinct classes of MTs with different dynamic properties. To understand how cellular proteins regulate the dynamics of MTs, it is necessary to have methods to assess their turnover properties. In this chapter we present approaches to assess MT dynamics in cultured mammalian cells using fluorescence redistribution after photobleaching. We include a discussion of cell culture and imaging conditions that maintain cell viability. We also provide an extensive discussion of both data collection and analysis that are utilized to estimate the turnover dynamics of MTs.
Methods in cell biology 01/2010; 97:35-52. · 1.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Within the mitotic spindle, there are multiple populations of microtubules with different turnover dynamics, but how these different dynamics are maintained is not fully understood. MCAK is a member of the kinesin-13 family of microtubule-destabilizing enzymes that is required for proper establishment and maintenance of the spindle. Using quantitative immunofluorescence and fluorescence recovery after photobleaching, we compared the differences in spindle organization caused by global suppression of microtubule dynamics, by treating cells with low levels of paclitaxel, versus specific perturbation of spindle microtubule subsets by MCAK inhibition. Paclitaxel treatment caused a disruption in spindle microtubule organization marked by a significant increase in microtubules near the poles and a reduction in K-fiber fluorescence intensity. This was correlated with a faster t(1/2) of both spindle and K-fiber microtubules. In contrast, MCAK inhibition caused a dramatic reorganization of spindle microtubules with a significant increase in astral microtubules and reduction in K-fiber fluorescence intensity, which correlated with a slower t(1/2) of K-fibers but no change in the t(1/2) of spindle microtubules. Our data support the model that MCAK perturbs spindle organization by acting preferentially on a subset of microtubules, and they support the overall hypothesis that microtubule dynamics is differentially regulated in the spindle.
Molecular biology of the cell 02/2009; 20(6):1639-51. · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advances in live-cell imaging technology have provided an unprecedented look at the dynamic behaviors of the plant microtubule cytoskeleton. Recent studies revisit the classic question of how plants create cell shape through the patterned construction of the cell wall. Visualization of the cellulose synthase complex traveling in the plasma membrane has brought a watershed of new information about cellulose deposition. Observation of the cellulose synthase complex tracking precisely over the underlying cortical microtubules has provided clear evidence that the microtubule array pattern serves as a spatial template for cellulose microfibril extrusion. Understanding how the microtubules are organized into specific array patterns remains a challenge, though new ideas are arising from genetic and cell biological studies. Long-term time-lapse observations of the microtubule arrays in light-grown hypocotyl cells have revealed a striking process of microtubule patterning possibly linked to the creation of polylamellate cell walls.
Current Opinion in Plant Biology 03/2008; 11(1):94-8. · 9.39 Impact Factor